Neurosteroids, steroids synthesized de novo in the brain, have been implicated in functions ranging from stress to depression, anxiety, and cognition. One neurosteroid is progesterone, a classic sex hormone involved in the regulation of reproduction. We have previously shown that peripheral estradiol (E2) increases hypothalamic neuroprogesterone (neuroP) synthesis, which initiates the luteinizing hormone (LH) surge. The LH surge triggers ovulation and the luteinization of the ruptured ovarian follicle - critical event in reproduction. This estrogen positive feedback is regulated by kisspeptin neurons of the hypothalamic rostral periventricular nucleus (RP3V). We propose to use immortalized, female, adult mouse hypothalamic kisspeptin neurons, mHypoA-51s, to examine the E2 and neuroP signaling underlying the LH surge. Preliminary data indicate that mHypoA-51 cell line is a good model for RP3V kisspeptin neurons: both have estrogen receptor-? (ER?), progesterone receptor (PR) and kisspeptin. Like native kisspeptin neurons, E2 in mHypoA- 51s induces expression of kisspeptin and PR, which is trafficked to the membrane. Surprisingly, subsequent stimulation of mHypoA-51s with P4 or astrocyte conditioned media induces calcium release and augments kisspeptin expression. Our overall hypothesis is that E2 induced PR mediates neuroP activation of kisspeptin neurons to induce the LH surge. Three sub-hypotheses will be tested in vitro and in vivo to characterize the integration of estrogenic and progestational signaling underlying estrogen positive feedback: (1) nuclear ER? induces PR, which is trafficked to the membrane and activated by neuroP; (2) neuroP activation of membrane localized PR stimulates kisspeptin release; and (3), neuroP signaling in kisspeptin neurons regulates the LH surge. Together the proposed studies will test a novel mechanism of progesterone signaling in the CNS, through which integration of peripheral and central steroidal information regulates reproduction.